p53 mutational spectra and the role of methylated CpG sequences

The occurrence of tumor-specific mutational spectra in the p53 mutation database provides indirect evidence that implicates certain exogenous and possibly endogenous mutagenic events in human carcinogenesis. In some cases, the distribution of DNA damage along the p53 gene caused by environmental carcinogens can be correlated with the mutational spectra, i.e. hotspots and types of mutations of certain cancers, most notably for nonmelanoma skin cancers and lung cancers in smokers. This concept has been validated by experiments with sunlight and cigarette smoke components representing the polycyclic aromatic hydrocarbon class of carcinogens. A disproportionally high number of mutations in p53 (and other genes) are found at methylated CpG dinucleotides. These sequences are particularly prone to mutagenesis involving endogenous events as well as modification by exogenous carcinogens.     

Wild type p53 reactivation: From lab bench to clinic

The p53 tumor suppressor is the most frequently inactivated gene in cancer. Several mouse models have demonstrated that the reconstitution of the p53 function suppresses the growth of established tumors. These facts, taken together, promote the idea of p53 reactivation as a strategy to combat cancer. This review will focus on recent advances in the development of small molecules which restore the function of wild type p53 by blocking its inhibitors Mdm2 and MdmX or their upstream regulators and discuss the impact of different p53 functions for tumor prevention and tumor eradication. Finally, the recent progress in p53 research will be analyzed concerning the role of p53 cofactors and cellular environment in the biological response upon p53 reactivation and how this can be applied in clinic.     

Mutations in p53 cDNA sequence introduced by retroviral vector

The high mutation rates of retroviruses are a potential problem with retroviral vectors. We studied the mutation rates and spectra of p53 sequences transduced with a retroviral vector in a cancer gene therapy model. When p53-deficient H358 non-small cell lung cancer cells were treated with a retroviral vector carrying normal p53 cDNA, most of transduced cells were killed by apoptosis. However, a small number of clones escaped p53-mediated apoptosis. We examined the p53 cDNA structure in these resistant clones. PCR-based analysis showed that 88/102 clones had detectable mutations in p53, including gross rearrangements, deletions/insertions, and base substitutions. To study the mutation rate of the p53 sequence in all transduced clones, the retroviral vector containing the non-functional p53 gene and the Neo-resistant marker gene was introduced into H358 cells. Only one of 95 isolated clones showed a base substitution. These results indicate that the mutation rate of p53 is not particularly high, but there is a significant risk that cancer cells will resist p53 gene therapy as a result of retroviral replication errors.     

Mutant p53 protein is targeted by arsenic for degradation and plays a role in arsenic-mediated growth suppression

p53 is frequently mutated in tumor cells, and mutant p53 is often highly expressed due to its increased half-life. Thus, targeting mutant p53 for degradation might be explored as a therapeutic strategy to manage tumors that are addicted to mutant p53 for survival. Arsenic trioxide, a drug for patients with acute promyelocytic leukemia, is found to target and degrade a class of proteins with high levels of cysteine residues and vicinal thiol groups, such as promyelocytic leukemia protein (PML) and PML-retinoic acid receptor α fusion protein. Interestingly, wild type p53 is accumulated in cells treated with arsenic compounds, presumably due to arsenic-induced oxidative stresses. In this study, we found that wild type p53 is induced by arsenic trioxide in tumor cells, consistent with published studies. In contrast, we found that arsenic compounds degrade both endogenous and ectopically expressed mutant p53 in time- and dose-dependent manners. We also found that arsenic trioxide decreases the stability of mutant p53 protein through a proteasomal pathway, and blockage of mutant p53 nuclear export can alleviate the arsenic-induced mutant p53 degradation. Furthermore, we found that knockdown of endogenous mutant p53 sensitizes, whereas ectopic expression of mutant p53 desensitizes, tumor cells to arsenic treatment. Taken together, we found that mutant p53 is a target of arsenic compounds, which provides an insight into exploring arsenic compound-based therapy for tumors harboring a mutant p53.     

Mutant p53 disrupts MCF-10A cell polarity in three-dimensional culture via epithelial-to-mesenchymal transitions

Mutant p53 is not only deficient in tumor suppression but also acquires additional activity, called gain of function. Mutant p53 gain of function is recapitulated in knock-in mice that carry one null allele and one mutant allele of the p53 gene. These knock-in mice develop aggressive tumors compared with p53-null mice. Recently, we and others showed that tumor cells carrying a mutant p53 are addicted to the mutant for cell survival and resistance to DNA damage. To further define mutant p53 gain of function, we used the MCF-10A three-dimensional model of mammary morphogenesis. MCF-10A cells in three-dimensional culture undergo a series of morphological changes and form polarized and growth-arrested spheroids with hollow lumen, which resembles normal glandular architectures in vivo. Here, we found that endogenous wild-type p53 in MCF-10A cells was not required for acinus formation, but knockdown of endogenous wild-type p53 (p53-KD) led to partial clearance of cells in the lumen due to decreased apoptosis. Consistent with this, p53-KD altered expression patterns of the cell adhesion molecule E-cadherin, the cytoskeletal marker β-catenin, and the extracellular matrix protein laminin V. We also found that ectopic expression of the mutant G245S led to a phenotype similar to p53-KD, whereas a combination of ectopic expression of siRNA-resistant G245S with p53-KD led to a less cleared lumen. In contrast, ectopic expression of mutant R248W, R175H, and R273H disrupted normal acinus architectures with filled lumen and led to formation of irregular and multiacinus structures regardless of p53-KD. In addition, these mutants altered normal expression patterns and/or levels of E-cadherin, β-catenin, laminin V, and tight junction marker ZO-1. Furthermore, epithelial-to-mesenchymal transitions (EMT) markers, Snail, Slug, and Twist, were highly induced by mutant p53 and/or p53-KD. Together, we postulate that EMT represents a mutant p53 gain of function and mutant p53 alters cell polarity via EMT.     

Survivin inhibits anti-growth effect of p53 activated by aurora B

Genomic instability and apoptosis evasion are hallmarks of cancer, but the molecular mechanisms governing these processes remain elusive. Here, we found that survivin, a member of the apoptosis-inhibiting gene family, and aurora B kinase, a chromosomal passenger protein, were co-overexpressed in the various glioblastoma cell lines and tumors. Notably, exogenous introduction of the aurora B in human BJ cells was shown to decrease cell growth and increase the senescence-associated beta-galactosidase activity by activation of p53 tumor suppressor. However, aurora B overexpression failed to inhibit cell proliferation in BJ and U87MG cells transduced with dominant-negative p53 as well as in p53(-/-) mouse astrocytes. Aurora B was shown to increase centrosome amplification in the p53(-/-) astrocytes. Survivin was shown to induce anchorage-independent growth and inhibit anti-proliferation and drug-sensitive apoptosis caused by aurora B. Overexpression of both survivin and aurora B further accelerated the proliferation of BJ cells. Taken together, the present study indicates that survivin should accelerate tumorigenesis by inhibiting the anti-proliferative effect of p53 tumor suppressor that is activated by aurora B in normal and glioblastoma cells containing intact p53.     

New approaches to understanding p53 gene tumor mutation spectra

The first p53 gene mutation arising in a human tumor was described a decade ago by Baker et al. [S.J. Baker, E.R. Fearon, J.M. Nigro, S.R. Hamilton, A.C. Preisinger, J.M. Jessup, P. van Tuinen, D.H. Ledbetter, D.F. Barker, Y. Nakamura, R. White, B. Vogelstein, Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas, Science 244 (1989) 217–221]. There are now over 10,000 mutations extracted from the published literature in the IARC database of human p53 tumor mutations [P. Hainaut, T. Hernandez, A. Robinson, P. Rodriguez-Tome, T. Flores, M. Hollstein, C.C. Harris, R. Montesano, IARC database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualization tools, Nucleic Acids Res. 26 (1998) 205–213; Version R3, January 1999]. A large and diverse collection of tumor mutations in cancer patients provides important information on the nature of environmental factors or biological processes that are important causes of human gene mutation, since xenobiotic mutagens as well as endogenous mechanisms of genetic change produce characteristic types of patterns in target DNA [J.H. Miller, Mutational specificity in bacteria, Annu. Rev. Genet. 17 (1983) 215–238; T. Lindahl, Instability and decay of the primary structure of DNA, Nature 362 (1993) 709–715; S.P. Hussain, C.C. Harris, Molecular epidemiology of human cancer: contribution of mutation spectra studies of tumor suppressor genes, Cancer Res. 58 (1998) 4023–4037; P. Hainaut, M. Hollstein, p53 and human cancer: the first ten thousand mutations, Adv. Cancer Res. 2000]. P53 gene mutations in cancers can be compared to point mutation spectra at the HPRT locus of human lymphocytes from patients or healthy individuals with known exposure histories, and accumulated data indicate that mutation patterns at the two loci share certain general features.

Hypotheses regarding specific cancer risk factors can be tested by comparing p53 tumor mutations typical of a defined patient group against mutations generated experimentally in rodents or in prokaryotic and eukaryotic cells in vitro. Refinements of this approach to hypothesis testing are being explored that employ human p53 sequences introduced artificially into experimental organisms used in laboratory mutagenesis assays. P53-specific laboratory models, combined with DNA microchips designed for high through-put mutation screening promise to unmask information currently hidden in the compilation of human tumor p53 mutations.     

穿心莲内酯恢复突变型p53野生型功能的作用及机制研究

目的 p53是人体内重要的肿瘤抑制因子,但在人类肿瘤中因高频突变而失去抑癌功能。突变型p53 (mutant p53,mutp53)可促进肿瘤的发生、发展和转移。由于在肿瘤细胞中通常有较高表达,mutp53已成为区别于正常细胞的一个特异性抗肿瘤靶点。本研究旨在探索穿心莲内酯的抗肿瘤作用机制,为寻找靶向mutp53的抗肿瘤化合物提供理论依据。方法 构建可以快速筛选具有恢复mutp53下游转录因子的荧光素酶系统,观察穿心莲内酯对H1299-p53 R273H-PUMAluciferase和H1299-p53R175H-PUMA-luciferase细胞中PUMA基因的表达情况;采用免疫荧光实验,检测穿心莲内酯对HT29(R273H)和SK-BR-3 (R175H)细胞中mutp53的表达影响;采用免疫印迹实验进一步观察穿心莲内酯恢复了mutp53肿瘤细胞中p53下游靶蛋白PUMA、p21、Noxa的表达;随后采用MTT和流式细胞分析,检测穿心莲内酯对肿瘤细胞增殖和凋亡的影响;此外,还通过si RNA敲低Hsp70表达后,研究穿心莲内酯对mutp53下游基因的重激活作用。结果 穿心莲内酯可以...     

Decision making of the p53 network: Death by integration

The tumor suppressor protein p53 plays a central role in the multiple response pathways activated by DNA damage. In particular, p53 is involved in both the pro-survival response of cell cycle arrest and DNA repair, and the pro-death response of apoptosis. How does the p53 network coordinate the different pathways that lead to the opposite cell fates and what is its strategy in making the life-death decisions? To address these questions, we develop an integrated mathematical model that embraces three key modules of the p53 network: p53 core regulation, p53-induced cell cycle arrest and p53-dependent apoptosis initiation. Our analyses reveal that different aspects of the nuclear p53 dynamic profile are being used to differentially regulate the pro-survival and the pro-death modules. While the activation of the pro-survival module is dependent on the current or recent status of the DNA damage, the activation of the pro-death module relies on the accumulation or integration of the damage level over time. Thus, the cell will take the death fate if it cannot recover from the damage within a time period that is inversely proportional to the damage level. This “adaptive timer” strategy is likely to be adopted in other stress response systems.     

Determination of sensitive positions to mutations in human p53 protein

Over last several years, we demonstrated that the mutations are more likely to occur at randomly unpredictable amino acid pairs in a protein. We therefore can in principle predict the amino acid pairs sensitive to the future mutations in a protein. However, we still need to predict the positions at which the sensitive amino acid pairs are located in a protein. In this study, we use a probabilistic approach to analyze the effect of 191 mutations in human p53 protein and can approximately estimate the sensitive positions to mutations in human p53 protein.     

New tricks of an old molecule: lifespan regulation by p53

As guardian of the genome the tumor suppressor p53 controls a crucial point in protection from cellular damage and response to stressors. Activation of p53 can have beneficial (DNA repair) or detrimental (apoptosis) consequences for individual cells. In either case activation of p53 is thought to safeguard the organism at large from the deleterious effects of various stresses. Recent data suggest that the function of p53 might also play a role in the regulation of organismal lifespan. Increased p53 activity leads to lifespan shortening in mice, while apparent reduction of p53 activity in flies leads to lifespan extension. Although the mechanism by which p53 regulates lifespan remains to be determined, these findings highlight the possibility that careful manipulation of p53 activity during adult life may result in beneficial effects on healthy lifespan.     

Selenium activates p53 and p38 pathways and induces caspase-independent cell death in cervical cancer cells

The mechanisms of sodium selenite-induced cell death in cervical carcinoma cells were studied during 24 h of exposure in the HeLa Hep-2 cell line. Selenite at the employed concentrations of 5 and 50 μmol/L produced time- and dose-dependent suppression of DNA synthesis and induced DNA damage which resulted in phosphorylation of histone H2A.X. These effects were influenced by pretreatment of cells with the SOD/catalase mimetic MnTMPyP or glutathione-depleting buthionine sulfoximine, suggesting the significant role of selenite-generated oxidative stress. Following the DNA damage, selenite activated p53-dependent pathway as evidenced by the appearance of phosphorylated p53 and accumulation of p21 in the treated cells. Concomitantly, selenite activated p38 pathway but its effect on JNK was very weak. p53- and p38-dependent signaling led to the accumulation of Bax protein, which was preventable by specific inhibitors of p38 (SB 203580) and p53 (Pifithrin-α). Mitochondria in selenite-treated cells changed their dynamics (shape and localization) and released AIF and Smac/Diablo, which initiated caspase-independent apoptosis as confirmed by the caspase-3 activity assay and the low effect of caspase inhibitors z-DEVD-fmk and z-VAD-fmk on cell death. We conclude that selenite induces caspase-independent apoptosis in cervical carcinoma cells mostly by oxidative stress-mediated activation of p53 and p38 pathways, but other selenite-mediated effects, in particular mitochondria-specific ones, are also involved.     

BclxL Changes Conformation upon Binding to Wild-type but Not Mutant p53 DNA Binding Domain

p53 can induce apoptosis through mitochondrial membrane permeabilization by interaction of its DNA binding region with the anti-apoptotic proteins BclxL and Bcl2. However, little is known about the action of p53 at the mitochondria in molecular detail. By using NMR spectroscopy and fluorescence polarization we characterized the binding of wild-type and mutant p53 DNA binding domains to BclxL and show that the wild-type p53 DNA binding domain leads to structural changes in the BH3 binding region of BclxL, whereas mutants fail to induce such effects due to reduced affinity. This was probed by induced chemical shift and residual dipolar coupling data. These data imply that p53 partly achieves its pro-apoptotic function at the mitochondria by facilitating interaction between BclxL and BH3-only proteins in an allosteric mode of action. Furthermore, we characterize for the first time the binding behavior of Pifithrin-μ, a specific small molecule inhibitor of the p53-BclxL interaction, and present a structural model of the protein-ligand complex. A rather unusual behavior is revealed whereby Pifithrin-μ binds to both sides of the protein-protein complex. These data should facilitate the rational design of more potent specific BclxL-p53 inhibitors.     

Susceptibility to cell death induced by blockade of MAPK pathway in human colorectal cancer cells carrying Ras mutations is dependent on p53 status

Constitutive activation of mitogen-activated protein kinase (MAPK) pathway is implicated in a variety of human malignancies especially those that carry Ras mutations and is currently exploited as a cancer therapeutic target. The variability of response by cancer cells to the inhibition of the Ras/MAPK pathway both in vivo and in vitro, however, suggests that the genetic background of the tumor cell may modulate the effectiveness of this directed therapeutic. In a panel of colorectal cancer cell lines that carry Ras mutations and have constitutively active MEK/MAPK, we found that inhibition of the MAPK upstream kinase MEK by the small molecular MEK inhibitor U0126 induced cell death only in p53 wild-type cells. By contrast, p53-deficient cells were not affected by blocking the MEK/MAPK pathway. Using isogenic colon cancer cell lines and RNA interference, we show that loss of p53 significantly reduces MAPK phosphorylation and renders cells resistant to U0126 treatment. These findings reveal a critical role for p53 in MAPK-driven cell survival and place p53 upstream in the control cascade of MAPK activity. The therapeutic implication of these observations is that MAPK inhibitors will be most beneficial as a therapeutic agent in p53 normal colon cancers where constitutively active MAPK resulting from a Ras mutation is required for cell survival.     

ik3-2, a relative to ik3-1/Cables, is involved in both p53-mediated and p53-independent apoptotic pathways

ik3-2 is a close relative to ik3-1/Cables, an associator with cdk3 and cdk5. ik3-1/Cables has been identified to be a candidate tumor suppressor for colon and head/neck cancers. In agreement, it has been pointed out that ik3-1/Cables is a regulator for both p53- and p73-induced apoptosis [J. Biol. Chem. 277 (2002) 2951] although ectopic expression of ik3-1/Cables does not induce apoptosis. Here we show that adenovirus-mediated overexpression of ik3-2 results in apoptosis of p53-intact U2OS cells. ik3-2 binds to p53 in vivo and ectopic coexpression of ik3-2 enhances apoptosis induced by adenovirus-mediated expression of p53. Furthermore, ectopic expression of ik3-2 results in apoptosis of primary p53/Mdm2- and p53/ARF-null mouse embryo fibroblasts, indicating that ik3-2-induced apoptosis is partially p53-independent. Both the highly conserved C-terminal cyclin box-homologous domain (ik3-2-C) and the N-terminal region consisting of 70 amino acids (ik3-2-N) are responsible for ik3-2-mediated enhancement of p53-induced apoptosis. In contrast, ik3-2-induced p53-independent apoptosis is mediated through ik3-2-N. We thus identified ik3-2 as a proapoptotic factor involved in both p53-mediated and p53-independent apoptotic pathways.     

Molecular dynamics of the full-length p53 monomer

The p53 protein is frequently mutated in a very large proportion of human tumors, where it seems to acquire gain-of-function activity that facilitates tumor onset and progression. A possible mechanism is the ability of mutant p53 proteins to physically interact with other proteins, including members of the same family, namely p63 and p73, inactivating their function. Assuming that this interaction might occurs at the level of the monomer, to investigate the molecular basis for this interaction, here, we sample the structural flexibility of the wild-type p53 monomeric protein. The results show a strong stability up to 850 ns in the DNA binding domain, with major flexibility in the N-terminal transactivations domains (TAD1 and TAD2) as well as in the C-terminal region (tetramerization domain). Several stable hydrogen bonds have been detected between N-terminal or C-terminal and DNA binding domain, and also between N-terminal and C-terminal. Essential dynamics analysis highlights strongly correlated movements involving TAD1 and the proline-rich region in the N-terminal domain, the tetramerization region in the C-terminal domain; Lys120 in the DNA binding region. The herein presented model is a starting point for further investigation of the whole protein tetramer as well as of its mutants.